Proteases are used in various applications, including pharmaceutical compositions such as digestive enzyme agents for alleviating gastrointestinal disorders, thrombolytic agents, or anti-inflammatory agents, and compositions for clothes, contact lenses or cleaners, as well as cosmetics, leather processing agents, food softeners, meat enhancers, feed of food additives, oil and fat separating agents, wastewater treatment, etc. The utility of enzymes among microbial products is already widely known. Proteases account for the highest percentage (60% or higher) of the industrial enzyme market, and the marketability thereof is more and more increasing.
About 25% of industrial proteases are marketed as detergents, and proteases for detergents are required to have a wide spectrum of substrate specificity capable of degrading food, blood or body fluid components, and an alkaline environment, and should be stable so that they do not lose their surfactant activity and enzymatic activity at high temperatures or low temperatures. In the past, plant-derived proteases were mainly used, but in recent years, microorganisms have been most frequently used to produce proteases.
If proteases are to be used for industrial applications, they are required to be very stable. For example, the activity of most proteins decreases or completely disappears in the presence of surfactants, and for this reason, proteases that are used in detergents are extremely limited. Also, if detergents are used at high concentrations, the activity of proteases contained therein is difficult to expect. In addition, conditions such as exposure to extreme pH, exposure to heavy metals, or the degree of oxidation-reduction, all strongly influence the activity of enzymes, and thus if these conditions are out of suitable ranges, enzymes rapidly lose their activity. For this reason, in order for proteases to be regularly used for industrial applications, the proteases are required to maintain their activity even under extreme and unstable physical and chemical conditions.
Serine-based proteases such as subtilisin have been most widely used in the detergent industry. Such basic proteases securely maintain their activity even under high pH conditions in which a surfactant and an oxidizing agent are present (Gupta et al., Appl Microbiol Biotechnol, 59:15-32, 2002; Haddar et al., Bioresour Technol, 100:3366-3373, 2009), and thus are useful in various industrial applications.
In recent years, a need for cold-active enzymes has increased. Particularly, low-active proteases have been added to detergents, and maintain their high activity at a laundry temperature of 15° C. or lower.
In documents regarding cold-adapted proteases, reported to date, there are reports of the cloning of serine-based cold-active protease genes derived from the psychrophilic microorganism Shewanella, the purification and characterization of enzymes (Kulakova et al., Appl Environ Microbiol, 65:611-617, 1999), the purification, characterization and sequencing (Huston et al., Appl Environ Microbiol, 70:3321-3328, 2004) of the psychrophilic marine microorganism Colwellia psychrerythraea, the purification and characterization of cold-adapted serine-based proteases derived from Colwellia sp., (Wang et al., Biotechnol Lett, 27:1195-1198, 2005) and the like.
Accordingly, the present inventors have made extensive efforts to develop proteases, which are used in various industrial applications, by using a cold-adapted and basic serine-based protease produced from Pseudoalteromonas arctica PAMC 21717 isolated from Antarctic Ocean sediments. As a result, the present inventors have isolated a cold-adapted protease that exhibits enzymatic activity under the conditions of 0 to 60° C. and pH 5.0 to 11.0 and then have identified the crystalline structure of the cold-adapted protease, and have found that the cold-adapted protease exhibits high activity at relatively low temperatures compared to proteases such as subtilisin, thereby completing the present invention.